Project Summary Mechanical forces are a key component of the cellular microenvironment, and are well established to have potent effects on cells and tissues. The passive mechanical properties of two-dimensional cell substrates and three-dimensional extracellular matrices have been shown to influence progenitor cell phenotype and can be used to direct cell function. In addition, active stimulation of cells and tissues using externally applied forces has been applied at both the cell and tissue level to induce a variety of responses. Mechanobiology is particularly relevant to musculoskeletal tissues, but there is a gap in our understanding of the physical properties of the tissue environment on length scales that cells sense. This project builds on preliminary work by the project team in applying advanced ultrasound techniques to studying the microscale physical properties of engineered musculoskeletal tissues composed of cell-seeded mineralizing hydrogels. It integrates spectral ultrasound imaging (SUSI), dual-mode ultrasound elastography (DUE), and ultrasound-induced compressive stimulation. SUSI is a technique that uses the backscattered radiofrequency spectrum to derive information about the composition of a sample. DUE applies acoustic radiation force to deform hydrogels and measure their mechanical properties. Focused ultrasound-induced compression also applies acoustic pressure to mechanically stimulate tissues. A key feature of ultrasound techniques is that they are noninvasive and therefore can be used to study developing tissues over time. In addition, imaging and deformation can be applied at sub-millimeter resolution. This project will combine these advanced ultrasound techniques to create a system that can comprehensively characterize and stimulate engineered musculoskeletal tissues at the microscale. The target application is to potentiate bone formation using mesenchymal stem cells (MSC) embedded in a 3D hydrogel matrix. The Specific Aims are 1) to integrate spectral ultrasound imaging (SUSI) and dual-mode ultrasound elastography (DUE) to compositionally and mechanically characterize mineralizing tissues, 2) to probe the effects of passive matrix mechanical properties on MSC phenotype using SUSI-DUE, 3) to actively stimulate osteogenic differentiation of MSC in hydrogel matrices using ultrasound-induced cyclic compression, and 4) to apply SUSI-DUE to catalyze and monitor bone regeneration in vivo. This project will investigate musculoskeletal mechanobiology using an innovative new tool that could have important impact on regenerative medicine. The long term goal is a therapeutic intervention to potentiate bone formation in indications where accelerated healing would lead to improved outcomes, such as treatment of non-unions and recalcitrant spinal fusions.